Identification of tumor initiating cells and early marker genes in normal colonic epithelium that lead to neoplastic transformation

Identification of tumor initiating cells and early marker genes in normal colonic epithelium that lead to neoplastic transformation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification of tumor initiating cells and early marker genes in normal colonic epithelium that lead to neoplastic transformation Sangeeta Jaiswal, Stephanie The, Tse-Shao Chang, Jiaqi Shi, Thomas D Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7914753/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background & Aims : Colorectal cancer (CRC) remains a major cause of cancer-related morbidity and mortality worldwide. Although the adenoma-carcinoma sequence and associated genetic alterations are well characterized, the earliest cellular and molecular events that initiate tumorigenesis within histologically normal colonic epithelium remain poorly defined. This study aims to identify tumor-initiating cells (TICs) and early transcriptional markers of neoplastic transformation using single-cell RNA sequencing (scRNA-seq) from paired normal-appearing and transformed human colonic tissues. Methods : Fresh biopsies from histologically normal-appearing colonic mucosa and paired polyps, including tubular adenomas, sessile serrated adenomas, and adenocarcinoma, were collected from 7 human subjects. Single-cell transcriptomes were generated using the 10X Genomics platform and analyzed with Seurat, Monocle 2, CytoTRACE, GSVA, GSEA, RNA velocity, and InferCNV. Tumor-associated states were inferred utilizing clustering, trajectory analysis, pathway enrichment, copy number variation profiling, and validated spatially by RNA-FISH. Results : A total of 51,054 high-quality single-cell transcriptomes were resolved into 33 epithelial and stromal clusters. Tumor-specific stem-like (tSTM, cluster 0) and deep crypt secretory (tDCS, cluster 10) populations were enriched in adenomas, whereas sub-clustering of tSTM identified TICs (subclusters 4 and 6) derived largely from histologically normal mucosa. TICs exhibited strong stemness potential, genomic instability, and early activation of epithelial-mesenchymal transition (EMT) and interferon signaling, accompanied by suppression of oxidative phosphorylation. ETS2 , SLC12A2 , and LEFTY1 were identified as TIC-specific markers, while SOD3 and GPRC5A showed progressive upregulation along the TIC-to-tSTM trajectory. RNA-FISH confirmed spatial expression of candidate genes in adenomatous crypts, and independent validation using the COLON MAP dataset supported the presence and diagnostic performance of TIC-associated markers. Conclusions : This study identifies TICs as the developmental origin of neoplastic stem-like states and delineates early transcriptional and pathway reprogramming events that drive the transition from normal to premalignant colonic epithelium. These findings provide new insight into CRC initiation and nominate biomarkers with translational potential for early detection and therapeutic targeting. Computational Biology Cancer Biology tumor-initiating cells colorectal cancer single-cell RNA-seq epithelial-to-mesenchymal transition ETS2 SOD3 GPRC5A Full Text Additional Declarations The authors declare no competing interests. Supplementary Files TableS1.xlsx TableS2.csv TableS3.xlsx TableS4.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7914753","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":534428479,"identity":"bacfcfa9-a808-4235-b809-9d5eb510fc20","order_by":0,"name":"Sangeeta Jaiswal","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Sangeeta","middleName":"","lastName":"Jaiswal","suffix":""},{"id":534428480,"identity":"6edcb3bd-4491-4ad4-989f-535fd36f4db9","order_by":1,"name":"Stephanie The","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"","lastName":"The","suffix":""},{"id":534428792,"identity":"0c3e8b27-509e-45e2-ba96-bcbf92fd62f9","order_by":2,"name":"Tse-Shao Chang","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Tse-Shao","middleName":"","lastName":"Chang","suffix":""},{"id":534428793,"identity":"06dbe938-d9a5-4dbd-a01a-5b6785dd5a0b","order_by":3,"name":"Jiaqi Shi","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"Shi","suffix":""},{"id":534428794,"identity":"11729c7c-d04f-4a37-95fc-3aff1ffa0029","order_by":4,"name":"Thomas D Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYDACCSjND8QHGBtAJAEdPDAtkg0kazEAqSRKi71089ENP/4clje+kZ144OcOBjm+GwkEbJE5lnazt+2w4bYbuRsO9p5hMJYkqEUix+wGb8NtRpCWw4xtDIkbiNFy88+f2/abZ0C01BOl5TYP2+3EDRIQLQkGBLXcSEu7Ldv2P3nGmbdAv7RJGM488wC/FvYZycduvvmTZtvfnrv5w882G3m+4wRsQQcShJWMglEwCkbBKCAMAEtFT+4rWxgQAAAAAElFTkSuQmCC","orcid":"","institution":"University of Michigan","correspondingAuthor":true,"prefix":"","firstName":"Thomas","middleName":"D","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-10-21 13:33:57","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7914753/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7914753/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94451333,"identity":"982e5d33-6531-40d5-89dc-228f683269c1","added_by":"auto","created_at":"2025-10-27 14:40:02","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5411295,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/037adb32a2d3de64057bec00.docx"},{"id":94450424,"identity":"b730ee99-e47a-4fd0-965a-87343a4f0e4b","added_by":"auto","created_at":"2025-10-27 14:39:16","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":342,"visible":true,"origin":"","legend":"","description":"","filename":"rs7914753.json","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/7e44205ee14cf9e31525027a.json"},{"id":94451188,"identity":"3b35cc3c-6808-462e-86e3-21a39e26e232","added_by":"auto","created_at":"2025-10-27 14:39:56","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155902,"visible":true,"origin":"","legend":"","description":"","filename":"rs79147530enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/56f583d4e58f778fbe581ff2.xml"},{"id":94450690,"identity":"425f67c8-6fdb-4ba3-a2ee-4a2e9a1460c5","added_by":"auto","created_at":"2025-10-27 14:39:32","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1021709,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/dc2d057a71100818d0391895.jpeg"},{"id":94451495,"identity":"3b9e06e0-b2be-4530-9d1e-c25215504fe1","added_by":"auto","created_at":"2025-10-27 14:40:11","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":959427,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/7c72a108c823f4fd8917d7f9.jpeg"},{"id":94451503,"identity":"01ef7f6c-d511-43f3-bb81-75869a9bfabe","added_by":"auto","created_at":"2025-10-27 14:40:11","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1578591,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/56bf03e67bcf65b043026e25.jpeg"},{"id":94451212,"identity":"65fb8de9-ecc7-453c-9459-5fc503721db6","added_by":"auto","created_at":"2025-10-27 14:39:57","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1137666,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage12.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/1fd43030744423b441d4c83f.jpeg"},{"id":94450684,"identity":"1d619c46-4ae1-4461-9a5b-b30350c15c26","added_by":"auto","created_at":"2025-10-27 14:39:31","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":660579,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/135aea5063522b23f9d61b2d.jpeg"},{"id":94451416,"identity":"de1bf17d-880c-42b1-a1c3-89fb6cd8f875","added_by":"auto","created_at":"2025-10-27 14:40:06","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":853685,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/9109bd6c5d70fec3c5fa3228.jpeg"},{"id":94451398,"identity":"05a9a6a0-b1e4-48c2-9ae3-0e501bf09bdd","added_by":"auto","created_at":"2025-10-27 14:40:06","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1229482,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/d04c7a751f76af636d8ba9c6.jpeg"},{"id":94450253,"identity":"224c6606-f4dc-4dea-a1dc-a076ce848697","added_by":"auto","created_at":"2025-10-27 14:39:09","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":849560,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/463b59b911f23fd5a9cd2bdb.jpeg"},{"id":94450652,"identity":"dca8b474-adba-463f-8639-5b3ce02e140a","added_by":"auto","created_at":"2025-10-27 14:39:28","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":539982,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/2c291e2ede1423c8bd681da7.jpeg"},{"id":94451622,"identity":"f8a3e34d-8000-4851-9614-f495e9aa0ee0","added_by":"auto","created_at":"2025-10-27 14:40:15","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2539668,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/cf3a309fbb358e5bbb7c9d55.jpeg"},{"id":94451406,"identity":"dc3b675e-44d9-43f2-b965-e64f053871f5","added_by":"auto","created_at":"2025-10-27 14:40:06","extension":"jpeg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":891963,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/d5ca927b2f43a32ba64e6a5f.jpeg"},{"id":94450763,"identity":"c5e72a86-090e-4ad3-a83e-f7c8a6a10658","added_by":"auto","created_at":"2025-10-27 14:39:35","extension":"jpeg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1576066,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/e2dff17fecb28033dd316eba.jpeg"},{"id":94451422,"identity":"3a10c076-a518-4a28-9f8e-f05511dd0b74","added_by":"auto","created_at":"2025-10-27 14:40:07","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":236310,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/a8268b38c9750acfd457eb44.png"},{"id":94451350,"identity":"af360430-0cea-422b-828b-0cab5f804c33","added_by":"auto","created_at":"2025-10-27 14:40:04","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":186643,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/35ab6fce36f6fa9930089259.png"},{"id":94451814,"identity":"2f529a97-04d8-45bd-bb37-d9a898cc7876","added_by":"auto","created_at":"2025-10-27 14:40:25","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":376031,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/29565d4b696f4914529e51be.png"},{"id":94451067,"identity":"50ae0882-7303-4a7c-a42a-caa4ce7f3174","added_by":"auto","created_at":"2025-10-27 14:39:47","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":266103,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/6b444fa1a3fb45ad9a24f703.png"},{"id":94451059,"identity":"df79651b-33e3-4a5f-8902-511e534afc15","added_by":"auto","created_at":"2025-10-27 14:39:46","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":194882,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/fe711e3ee98ba59b653d1709.png"},{"id":94450645,"identity":"2d0e54fe-9d90-4be5-9d01-0294088a7ba8","added_by":"auto","created_at":"2025-10-27 14:39:27","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":240359,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/cb4616fbd774a226d32936d8.png"},{"id":94450648,"identity":"b1ac63aa-f4b2-4ee2-be27-a3483f10523a","added_by":"auto","created_at":"2025-10-27 14:39:27","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":345627,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/92316c6af5f131a0fc009e96.png"},{"id":94450254,"identity":"af1d4245-5ede-48ae-846e-fc20e50c97f6","added_by":"auto","created_at":"2025-10-27 14:39:09","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":174874,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/583eb4581d3a3f739714d57d.png"},{"id":94451106,"identity":"a0e58f8b-4848-46ed-ab00-36f744066eac","added_by":"auto","created_at":"2025-10-27 14:39:50","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126614,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/fa8d7c7639c76ec445ba6fb0.png"},{"id":94450657,"identity":"3ec3bd83-0e3e-4aca-ba5a-b36bc20032cc","added_by":"auto","created_at":"2025-10-27 14:39:29","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":993633,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/3d2369e98ec8e1c66892c1d5.png"},{"id":94450335,"identity":"83032840-2f81-4d09-991e-16639d3ab757","added_by":"auto","created_at":"2025-10-27 14:39:11","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":301559,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/477ca6c6fbd6ab2cf105f876.png"},{"id":94451427,"identity":"9b4c3146-1232-4cee-8cc0-37e8d48bdf9e","added_by":"auto","created_at":"2025-10-27 14:40:07","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":462736,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/a7d59797edd043f01b1487f5.png"},{"id":94451786,"identity":"92acbd5a-a4f1-48d5-90ba-c49ae7fe8aed","added_by":"auto","created_at":"2025-10-27 14:40:22","extension":"xml","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":153313,"visible":true,"origin":"","legend":"","description":"","filename":"rs79147530structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/4ed41e2344510ff884567724.xml"},{"id":94451332,"identity":"80c11806-fa8b-49ac-a677-5e7cdbb11961","added_by":"auto","created_at":"2025-10-27 14:40:02","extension":"html","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":169720,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/c58bfaae9752d72d3963508f.html"},{"id":94467556,"identity":"f5bd4119-2946-49ef-820b-292467773643","added_by":"auto","created_at":"2025-10-27 15:23:05","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2974923,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1_covered_c3522e9f-9de2-46f0-9a98-f5185387b345.pdf"},{"id":94451497,"identity":"ad09cf15-5518-41ed-9617-63fe684ba75f","added_by":"auto","created_at":"2025-10-27 14:40:11","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9412,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/c9deb492150795955d5c24f7.xlsx"},{"id":94450649,"identity":"95fea66b-d09f-4376-8bb3-ec637a8cd25c","added_by":"auto","created_at":"2025-10-27 14:39:27","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2652585,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.csv","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/bb3098c7c7bfed30c3d0ee72.csv"},{"id":94450719,"identity":"0da30a68-3b50-45e2-9949-0e519c03005f","added_by":"auto","created_at":"2025-10-27 14:39:33","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10117,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/c4aec29de0cc48848d2e9540.xlsx"},{"id":94451419,"identity":"38c13f2f-7563-401e-8ded-254b8c1a630d","added_by":"auto","created_at":"2025-10-27 14:40:07","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14352,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7914753/v1/45ec80c2c40fceb1da05e8bc.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eIdentification of tumor initiating cells and early marker genes in normal colonic epithelium that lead to neoplastic transformation\u003c/strong\u003e\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Michigan–Ann Arbor","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"tumor-initiating cells, colorectal cancer, single-cell RNA-seq, epithelial-to-mesenchymal transition, ETS2, SOD3, GPRC5A","lastPublishedDoi":"10.21203/rs.3.rs-7914753/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7914753/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u0026amp; Aims\u003c/strong\u003e: Colorectal cancer (CRC) remains a major cause of cancer-related morbidity and mortality worldwide. \u0026nbsp;Although the adenoma-carcinoma sequence and associated genetic alterations are well characterized, the earliest cellular and molecular events that initiate tumorigenesis within histologically normal colonic epithelium remain poorly defined. \u0026nbsp;This study aims to identify tumor-initiating cells (TICs) and early transcriptional markers of neoplastic transformation using single-cell RNA sequencing (scRNA-seq) from paired normal-appearing and transformed human colonic tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Fresh biopsies from histologically normal-appearing colonic mucosa and paired polyps, including tubular adenomas, sessile serrated adenomas, and adenocarcinoma, were collected from 7 human subjects. \u0026nbsp;Single-cell transcriptomes were generated using the 10X Genomics platform and analyzed with Seurat, Monocle 2, CytoTRACE, GSVA, GSEA, RNA velocity, and InferCNV. \u0026nbsp;Tumor-associated states were inferred utilizing clustering, trajectory analysis, pathway enrichment, copy number variation profiling, and validated spatially by RNA-FISH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: \u0026nbsp;A total of 51,054 high-quality single-cell transcriptomes were resolved into 33 epithelial and stromal clusters. \u0026nbsp;Tumor-specific stem-like (tSTM, cluster 0) and deep crypt secretory (tDCS, cluster 10) populations were enriched in adenomas, whereas sub-clustering of tSTM identified TICs (subclusters 4 and 6) derived largely from histologically normal mucosa. \u0026nbsp;TICs exhibited strong stemness potential, genomic instability, and early activation of epithelial-mesenchymal transition (EMT) and interferon signaling, accompanied by suppression of oxidative phosphorylation. \u0026nbsp;\u003cem\u003eETS2\u003c/em\u003e, \u003cem\u003eSLC12A2\u003c/em\u003e, and \u003cem\u003eLEFTY1\u003c/em\u003e were identified as TIC-specific markers, while \u003cem\u003eSOD3\u003c/em\u003eand \u003cem\u003eGPRC5A\u003c/em\u003e showed progressive upregulation along the TIC-to-tSTM trajectory. \u0026nbsp;RNA-FISH confirmed spatial expression of candidate genes in adenomatous crypts, and independent validation using the COLON MAP dataset supported the presence and diagnostic performance of TIC-associated markers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: This study identifies TICs as the developmental origin of neoplastic stem-like states and delineates early transcriptional and pathway reprogramming events that drive the transition from normal to premalignant colonic epithelium. \u0026nbsp;These findings provide new insight into CRC initiation and nominate biomarkers with translational potential for early detection and therapeutic targeting.\u003c/p\u003e","manuscriptTitle":"Identification of tumor initiating cells and early marker genes in normal colonic epithelium that lead to neoplastic transformation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 11:32:25","doi":"10.21203/rs.3.rs-7914753/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3732e081-36d6-49f1-8b7f-df5c3fe6dcc4","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56659373,"name":"Computational Biology"},{"id":56659374,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2025-10-27T11:32:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-27 11:32:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7914753","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7914753","identity":"rs-7914753","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}